Metabolites of ginseng saponins by human intestinal bacteria and its preparation for an anticancer

ABSTRACT

This invention relates to ginsenoside Mc with formula (I), ##STR1## an intestinal flora metabolite of ginseng saponin and anticancer agent containing it as an active ingredient. In addition to a novel compound, the anticancer agent of this invention consists of one active ingredient elected from compound K, compound Y or 20(S)-protopanaxatriol, intestinal flora metabolites of ginseng saponin, together with one or more pharmaceutically acceptable carriers. Said agent is a novel type of potential anticancer agent since it has immunopotentiating actions including inhibitory actions on the vascularization of tumors and extravasation of cancer cells.

The present application is a 371 of PCT/KR96/00281, filed Dec. 31, 1996.

FIELD OF THE INVENTION

This invention relates to the metabolites of ginseng saponins by humanintestinal bacteria and its preparation for an anticancer and moreparticularly, to a novel saponin of a metabolite of Panax ginsengsaponin by human bacteria and a novel preparation of anticancer agentcontaining a novel saponin of a metabolite of Panax ginseng saponin byhuman bacteria, which exhibits immunopotentiating actions includinginhibitory actions on the vascularization of tumors and extravasation ofcancer cells.

DESCRIPTION OF THE RELATED ART

In recent years much of the development of novel anticancer agents haswidely focused on natural sources and synthetic compounds.

Among saponins extracted from Panax ginseng, for example, ginsenosideRh₂ 3-0-β-D-glucopyranosyl-20(s)-protopanaxadiol! was reported toinhibit the proliferation of liver cancer cells (reference: JapanesePatent No. 89-28759).

Further, both ginsenoside Rg₃ 3-0-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl!-20(R)-protopanaxadiol! andginsenoside Rb₂ 20-0- α-L-arabinopyranosyl(1→6)-β-D-glucopyranosyl-3-0-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl!-20(S)-protopanaxadio 1! werereported to inhibit the vascularization of tumors and extravasation ofcancer cells including inhibitory actions on the metastatis of cancercells References: Japanese Patent No. 93-9123, Sato et al.: Biol. Pharm.Bull., 17. 635(1994)!.

In case of 20-0-β-D-glucopyranosyl!-20(S)-protopanaxadiol, called as"compound K" and 20-0- α-L-arabinopyranosyl(1→6)-β-D-glucopyranosyl!-20(S)-protopanaxadiol, called as "compound Y",which were isolated from soil strains of Panax ginseng saponin andintestinal flora of rats, their structure was already determinedReferences: Yoshioka et al.: Chem. Pharm. Bull., 20, 2418(1972), Takinoet al.: Medicinal ginseng '69 (Public Publishing Co., Ltd. 267(1989)!.

Also, the structure of 20(S)-protopanaxatriol, isolated by sapogenin ofPanax ginseng saponin, was already established (Nagai et al.:Tetrahedron, 27, 881(1971)!.

The pharmacological actions of these compounds, for example, inhibitionof glucose transplant related to cancer cells by the blockage ofmembrane protein, were merely reported by each inventor Hasegawa et al.,Planta Med., 60, 197(1994)!, including the report onmethicillin-resistant bacteria and the inhibition of the excretion ofdrugs on the multidrugs-resistant cancer cells Hasegawa et al.:Phytother, Res., 9, 260(1996), Hasegawa et al., Planta Med., 61,409(1995)!.

In case of the conventional chemotherapeutics which exhibit theirtherapeutic effects by attacking the cancer cells directly, theiradverse effects are quite severe. During several decades, anyantineoplastic agents with new mode of mechanism have not yet to be onthe market. Further, in the event that Panax ginseng saponins areapplied for the treatment of some diseases, these substances arereported to be metabolized by intestinal bacteria and said bacteria isliable to be influenced by human's constitution and his food pattern.Thus, any individual differences in the metabolism of sanponins may leadto the individual differences in his treatment.

SUMMARY OF THE INVENTION

In view of these situations, the inventors of this invention haveinvestigated the metabolism of ginseng saponin associated by humanintestinal bacteria and succeeded in isolating and identifying thefollowing compounds, i.e., a) protopanaxadiol saponins(ginsenoside Rb₁,ginsenoside Rb₂ and ginsenoside Rc), b) compound K, compound Y and 20-0-α-L- arabinofuranosyl(1→6)-β-D-glucopyranosyl-20(S)-protopanaxadiol!,which are called as ginsenoside Mc. metabolites of ginsenoside Rd, andc) 20(S)-protopanaxatriol, a metabolite of ginsenoside Rg₁ andginsenoside Re which belongs to protopanaxatriol saponin.

The inventor ascertains that these intestinal flora metabolites areabsorbed from intestinal tracts to blood and excreted via urine andfeces. By assuring that said intestinal flora metabolites prove to bemain substances of Panax ginseng saponin, the inventor has endeavored todevelop the therapeutic dosage form containing the active ingredient ofsaponin, which is not influenced by the difference of intestinalbacteria. As a result of reviewing these physiological activities, theinventor has discovered a novel preparation of anticancer agent, whichexhibits immunopotentiating actions including inhibitory actions on thevascularization of tumors and extravasation of cancer cells. Thus, thisinvention has finally completed.

Therefore, the object of this invention is to provide a new compound of20-0-α-L-arabinofuranosyl(1→6)-β-D-glucopyranosyl-20(S)-protopanaxadiol!having the following formula, a novel ginseng saponin metabolite byhuman intestinal bacteria(called as ginsenoside Mc) with the followingcharacteristics.

1) Structural fomula ##STR2##

2) Molecular formula: C₄₁ H₇₀ O₁₂

3) The mass spectrum (Fab-MS, negative and m/z) showed signals at 753M-H!⁻, 621(M-arabinofuranose-H!⁻, and 459M-arabinofuranos-glucopyranos-H!⁻.

4) The ¹ H-NMR spectrum(d5-pyridine) showed signals at δ 3.39(1H, t,J=10.5, 5.1 Hz, H-3), 0.80(1H, d, J=11.0 Hz, H-5), 3.93(1H, ddd-like,H-12), 5.32(1H, t, J=7.1 Hz, H-24), 0.92(3H, s, Me-18), 0.89(3H, s,Me-19), 1.69(3H, s, Me-21), 1.67(3H, s, Me-26), 1.67(3H, s, Me-27),1.21(3H, s, Me-28), 1.01(3H, s, Me-29), 0.99(3H, s, Me-30), 5.10(1H, d,J=7.8 Hz, H-1'-20-glucopyranosyl), 5.61(1H, J=1.7 Hz,H-1"-6'-arabinofuranosyl).

5) The ¹³ C-NMR spectrum(d5-pyridine) for aglycon moiety showed signalsat δ39.5(C-1), 28.3(C-2), 78.2(C-3), 39.5(C-4), 56.5(C-5), 18.8(C-6),35.2(C-7), 40.2(C-8), 50.4(C-9), 37.4(C-10), 30.8(C-11), 70.3(C-12),49.5(C-13), 51.5(C-14), 30.9(C-15), 26.7(C-16), 51.8(C-17) 16.3(C-18),16.1(C-19), 83.2(C-20), 22.4(C-21), 36.2(C-22), 23.2(C-23), 126.1(C-24),131.0(C-25), 25.8(C-26), 17.9(C-27), 28.7(C-28), 16.4(C-29), 17.5(C-30),

The ¹³ C-NMR spectrum(d5-pyridine) for 20-glucopyranosyl moiety showedsignals at 98.1(C-1'), 75.1(C-2'), 79.2(C-3'), 72.2(C-4'), 76.5(C-5'),68.5(C-6'),

The ¹³ C-NMR spectrum(d5-pyridine) for 6'-arabinofuranosyl moiety showedsignals at 110(C-1"), 83.5(C-2"), 79.0(C-3"), 86.3(C-4"), 62.8(C-5").

In addition to ginsenoside compound Mc, a novel intestinal bacteriametabolite of novel ginseng saponin, another object of this invention isto provide an anticancer agent containing one active ingredient selectedfrom intestinal bacteria metabolites of ginseng saponin such as compoundK, compound Y, ginsenoside Mc and protopanaxatriol, together with one ormore pharmaceutically acceptable carriers. These intestinal bacteriametabolites of ginseng saponin exhibits remarkable antineoplasticeffects in the long run since they potentiate the inhibitory actionsagainst cancer cells in lymphocyte and inhibit the vascularization oftumors and extravasation of cancer cells.

Even though the severity of symptoms differs, the oral dose ofanticancer agent for adult according to this invention is 1-50 mg/60 kgof body weight once daily or several times per a day, preferably 3-15mg/day/60 kg of body weight.

The anticancer agent according to this invention contains either asingle ingredient, or said ingredient plus one or more pharmaceuticallyacceptable carriers such as excipients in the form of solid or liquid.

The administration method and available dosage forms are as follows;

a) Oral forms: powders, tablets, suspensions, emulsifiers, capsules,granules, troches, pills, suspensions, spirits, syrups and limonades;

b) Injectable forms, or

c) Topical forms: ointments, solids, suspensions, powders, paps,suppositories, aerosols, cataplasmas, liniments, lotions, enemas andemulsifiers.

According to this invention, well-known excipients in the form of solidor liquid may be used As mentioned in the above, the formulation shouldbe conducted so as to contain the active ingredient of this inventionnecessary for single dose. The several examples of excipients used inrelated dosage forms are as follows:

Excipients in powders and other oral powders: lactose, crystallinecellulose, starch, dextrin, calcium phosphate, calcium carbonate,synthetic and natural aluminum dioxide, magnesium oxide, dried aluminumhydroxide, magnesium stearate, and sodium bicarbonate;

Excipients in topical powders: zinc oxide, talc, starch, kaolin, boratepowder, zinc stearate, magnesium stearate, magnesium carbonate,precipitated calcium carbonate, bismuth subgallate, and potassiumaluminum sulfate powder;

Excipients in liquids: water, glycerin, propylene glycol, sweet-tastesyrup, ethanol, fatty oil, ethylene glycol, polyethylene glycol, andsorbitol;

Excipients in ointments: hydrophobic or hydrophilic base (includingoil-soluble base, water-soluble base and suspended base) prepared bymixing fat, fatty oil, lanoline, vaseline, glycerin, wax, Japan wax,paraffin, paraffin sulfate, resins, higher alcohols, plastics, glycols,water, or surfactant.

EXAMPLE Preparation of ginsenoside Mc

The suspended solution of human flora was precultured in GAM mediumovernight and then,100 mg of ginsenoside Rc was added to said medium tothe desired concentration of 2% in a newly sterile GAM medium and thencultured at 37° C. for 1 day. The medium was extracted by 1-butanol andthe extracted solution was concentrated and purified onreversed/irreverse phase chromatography to give 25 mg of pureginsenoside Mc.

Formulation example!

Formulation example 1: A mixture of lactose, crystalline cellulose and1% magnesium stearate was added to 30 mg of compound K, an intestinalflora metabolite of ginseng saponin, for homogenous mixing. Said mixturewas tabletted by a tabletting machine to obtain each tablet containing200 mg.

Formulation examples 2˜4: Based upon the same procedure as described informulation 1, each preparation was obtained containing 30 mg ofcompound Y, ginsenoside Mc or 20(S)-protopanaxatriol, respectively,instead of 30 mg of compound K.

Formulation example 5: A solution of 15 mg of compound K, an intestinalflora metabolite of ginseng saponin and polysorbit 80 was filled into asterilized vial aseptically and after removing the moisture, thepreparation for injection was obtained.

Formulation example 6˜8: Based upon the same procedure as described informulation 5, each preparation was obtained containing 15 mg ofcompound Y, ginsenoside Mc or 20(S)-protopanaxatriol, respectively,instead of 15 mg of compound K.

The physiological actions involved in the intestinal flora metabolite ofginseng saponin of this invention are described in the followingexamples but these protective scopes are not confined to said examples.

Experiment 1!

Antitumor activity on leukemia cell line (P388) in lymphocyte of mice

a) Experimental method

Spleen lymphocytes of mice and leukemia cell line (P388) were used forthis experiment. Spleen lymphocytes (4×10⁶ cells) and leukemia cells(P388) (2×10⁸ cells) were cultured in a medium (RPMI 1640 supplementedwith 20 μM mercaptoethanol and 10% fetal bovine serum) containingintestinal flora metabolite of ginseng saponin (2.5 μM) in 5% CO₂saturated with steam for 16 hours. Aside from this, same numbers ofspleen lymphocytes or leukemia cell line were cultured in a mediumcontaining intestinal flora metabolite of ginseng saponin in same isconcentration as a control. The number of each survived cell waspurified by MTT method to calculate impaired rate of cells on leukemiacells (P388) of lymphocytes.

b) Experimental results

As shown in table 1, the experimental results revealed that allintestinal flora metabolites of ginseng saponin in a low concentrationof 2.5 μM exhibited antitumor activities 1.6 to 2 times as higher ascontrol group.

                  TABLE 1    ______________________________________    Antitumor activity on the cancer cells of lymphocyte    by intestinal flora metabolites of ginseng saponin               Concentration (μg/ml)                           Antitumor activity (%)    ______________________________________    Control                    31.3     1    Compound K   1.56          51.5     1.6    Compound Y   1.86          56.6     1.8    Ginsenoside Mc                 1.86          63.3     2.0    20(S)-protopanaxatriol                 1.19          82.4     2.0    ______________________________________

Experiment 2!

Inhibition on the vascularization of tumor (Test for the inhibition onthe proliferation)

a) Experimental method

Human lymphocyte (HL), leukemia cell line (K562) and bovine arteryendotheliocyte (BAE) were used for this experiment. HL (1×10⁵ cells),leukemia cell line (K562) (2×10⁵ cells) and BAE (5×10³ cells) werecultured in a medium (HL, K562: RPMI 1640 medium containg 10% fetalbovine, BAE: DMEM medium containing 10% fetal bovine) containingintestinal flora metabolites of ginseng saponin concentrated with 2-folddilution in 5% CO₂ saturated with steam (HL, K562: 24 hours, BAE: 72hours). The number of each survived cell was purified by MTT method tocalculate 50% inhibition concentration (IC₅₀), impaired rate of cells(IC₅₀ (HL)/IC₅₀ (BAE) and IC₅₀ (K562)/IC₅₀ (BAE).

b) Experimental results

As shown in table 2, the experimental results revealed that ginsenoside,Mc and 20(S)-protopanaxatriol exhibited inhibitory activities on theproliferation of tumor cells

                  TABLE 2    ______________________________________    Inhibitory activity on the proliferation by    intestinal flora metabolites of ginseng saponin              IC.sub.50 (μM)                            IC.sub.50 (C)/IC.sub.50 (BAE)              HL    K562    BAE     C = HL K562    ______________________________________    Compound K   45     26      28    1.7    1.6    Compound Y   83     78      32    2.6    2.6    Ginsenoside Mc                220     480     26    8.5    18    20(S)-protopanaxatriol                280     49      36    7.8    1.4    ______________________________________

Experiment 3!

Inhibition on the vascularization of tumor (Test for the inhibition onthe migration)

a) Experimental method

Bovine artery endotheliocyte (BAE) was used for this experiment. BAE(5×10³ cells) was cultured in 6-well plate for 24 hours and cellsattached to the plate were detached by a razor. Said medium was replacedby a new one and after one hour, a solution of intestinal florametabolite of ginseng saponin was added to the desired inhibitoryconcentration of 10% and 50%, respectively for 24-hour cultivation.After completing the cultivation, cells were fixed with methanol,stained with Giemsa method and counted the cells migrated from thedetached line under microscope.

b) Experimental results

As shown in table 3, the experimental results revealed that eachintestinal metabolite of ginseng saponin exhibited an inhibitoryactivity of migration and among them, it was noted that compound Kshowed more potent inhibitory activity of migration than Suramin (WakoPure Chem Ind. Ltd., Japan), a control group.

                  TABLE 3    ______________________________________    Migration-inhibition by intestinal flora metabolites of ginseng saponin                  Migration-inhibition (% control)                  IC.sub.10                           IC.sub.50    ______________________________________    Suramin         -3.8       37.1    Compound K        4.6      43.2    Compound Y      -3.7       28.9    Ginsenoside Mc  -1.6       30.0    20(S)-protopanaxatriol                    -1.6       30.0    ______________________________________

Experiment 4!

Inhibition on the extravasation of basement membrane

a) Experimental method

The transwell culture chamber was used for this experiment with ahaptoinvasion method (reference: Cancer Res., 47, 3239, (1987)). Thelower side of a filter having a hole of 8.0 μm in diameter was coatedwith 5 μg of matrigel for the fabrication of matrigel/FN filter. Humanadenosarcoma tissue cell (HT1080), treated in the intestinal florametabolite of ginseng saponin in a concentration of 1-1000 μM at 37° C.for 30 minutes, was charged to the upper side of each filter with 1×10⁵cells/100 μL. Then, said filters were added to 24-well plate having 600μL of MEM medium supplemented with 0.1% bovine serum albumin and themcultured for 4-hour cultivation. After completing the cultivation, cellswere fixed with methanol and stained with hematoxylin, a tissue stainingagent. Following the removal of cells at the upper side with a cottonpole, cells infiltrating into the lower side were counted undermicroscope.

b) Experimental results

As shown in table 4, the experimental results revealed that eachintestinal metabolite of ginseng saponin exhibited more potentinhibitory activity of extravasation than RGDS peptide (underdevelopment from Glycomed Co.:Cancer Res., 49, 3815 (1989)), a controlgroup and among them, 50% extravasation-inhibitory concentration ofcompound K showed a potent activity in a low concentration of 3.2 μM.

                  TABLE 4    ______________________________________    Inhibition on the extravasation of basement membrane    by intestinal flora metabolites of ginseng saponin               Concentration                        No. of infiltrated                                    Inhibition               (μM)  cancer cell/field                                    rate (%)    ______________________________________    Control                 118 ± 8    RGDS peptide 4000       61 ± 9   48    Compound K     1        73 ± 4   38                 ED.sub.50 = 3.2        50                  10        45 ± 9   62                  100       0           100    Control                 117 ± 9    RGDS peptide 4000       51 ± 10  56    Compound Y     1        125 ± 7                  10         92 ± 11 21                 ED.sub.50 = 31         50                  100       24 ± 2   62                 1000       0           100    Control                 117 ± 9    RGDS peptide 4000        51 ± 10 56    ginsenoside Mc                   1        114 ± 12  3                 ED.sub.50 = 7.6        50                  10        44 ± 7   62                  100        3 ± 1   97                 1000       0           100    Control                 97 ± 8    RGDS peptide 4000       49 ± 4   49    20(S)-protopanaxatriol                   1        103 ± 14                  10        93 ± 7    4                 ED.sub.50 = 48         50                  100       18 ± 4   81                 1000        1 ± 1   99    ______________________________________

From the aforementioned results, it is noted that intestinal florametabolites of ginseng saponin, such as compound K, compound Y,20(S)-protopanaxatriol including ginsenoside Mc of this invention, arenovel types of potential anticancer agent since they haveimmunopotentiating actions including inhibitory actions on thevascularization of tumors and extravasation of cancer cells.

The toxicity of ginsenoside Mc, a novel compound of this invention, isnearly negligible in some animal experiments with rats and mice and theproducts' stability of each preparation based upon each formulationexample is quite effective.

What is claimed is:
 1. A compound of the formula: ##STR3## .
 2. Apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of claim 1 and a pharmaceutically acceptablecarrier.
 3. The pharmaceutical composition of claim 2, wherein theactive ingredient is administered in a dosage of between 1.0-50mg/Kg/day.
 4. The pharmaceutical composition of claim 3, wherein theform of the composition is selected from the group consisting of anorally administrable form, an injectable form, and an externallyapplicable form.
 5. The pharmaceutical composition of claim 4, whereinthe orally administrable form is selected from the group consisting of atablet, a powder, a suspension, an emulsion, a capsule, a granule, atroche, a pill, a liquid, a spirit, a syrup and a limonade.
 6. Thepharmaceutical composition of claim 4, wherein the injectable form isselected from the group consisting of a liquid, a suspension and asolution.
 7. The pharmaceutical composition of claim 4, wherein theexternally applicable form is selected from the group consisting of anointment, a liquid, a powder, a plaster, a suppository, an aerosol, aliniment, a lotion, an enema and an emulsion.
 8. A pharmaceuticalcomposition comprising a pharmaceutically effective amount of a compoundselected from the group consisting of 20-0-β-D-glucopyranosyl!-20(S)-protopanaxadiol (compound K), 20-0-α-L-arabinopyranosyl(1→6)-β-D-glycopyranosyl!-20(S)-protopanaxadiol(compound Y), and20(S)-protopanaxatriol, together with one or more pharmaceuticallyacceptable carriers.
 9. The pharmaceutical composition of claim 8,wherein the active ingredient is 20-0-β-D-glucopyranosyl!-20(S)-protopanaxadiol (compound K).
 10. Thepharmaceutical composition of claim 8, wherein the active ingredient is20-0-α-L-arabinopyranosyl(1→6)-β-D-glycopyranosyl!-20(S)-protopanaxadiol(compound Y).
 11. The pharmaceutical composition of claim 8, wherein theactive ingredient is 20(S)-protopanaxatriol.
 12. The pharmaceuticalcomposition of claim 8, wherein the active ingredient is administered ina dosage of between 1.0-50.0 mg/Kg/day.
 13. The pharmaceuticalcomposition of claim 12, wherein the form of the composition is selectedfrom the group consisting of an orally administrable form, an injectableform, and an externally applicable form.
 14. The pharmaceuticalcomposition of claim 13, wherein the orally administrable form isselected from the group consisting of a tablet, a powder, a suspension,an emulsion, a capsule, a granule, a troche, a pill, a liquid, a spirit,a syrup and a limonade.
 15. The pharmaceutical composition of claim 13,wherein the injectable form is selected from the group consisting of aliquid, a suspension and a solution.
 16. The pharmaceutical compositionof claim 13, wherein the externally applicable form is selected from thegroup consisting of an ointment, a liquid, a powder, a plaster, asuppository, an aerosol, a liniment, a lotion, an enema and an emulsion.